1. Field of the Invention
The present invention relates to a spectral measuring method of qualitatively or quantitatively analyzing a sample by irradiating the sample with excitation light of a single wavelength and detecting fluorescence or Raman scattered light which is generated from the sample, and a spectral measuring apparatus employed therefor.
2. Description of the Background Art
Optical analyzing methods include a method called a Raman spectrometry. When certain molecules are illuminated, a small percentage of the molecules which have retained a photon do not return to their original vibration level after remitting the retained photon, but drops to a different vibrational level of the ground electronic state. The radiation emitted from these molecules will therefore be at a different energy and hence a different wavelength. This is referred to as Raman scattering.
If the molecule drops to a higher vibrational level of the ground electronic state, the photon emitted is at a lower energy or longer wavelength than that retained. This is referred to as Stokes-shifted Raman scattering. If a molecule is already at a higher vibrational state before it retains a photon, it can impart this extra energy to the remitted photon thereby returning to the ground state. In this case, the radiation emitted is of higher energy (and shorter wavelength) and is called anti-Stokes-shifted Raman scattering. In any set of molecules under normal conditions, the number of molecules at ground state is always much greater than those at an excited state, so the odds of an incident photon hitting an excited molecule and being scattered with more energy than it carried upon collision is very small. Therefore, photon scattering at frequencies higher than that of the incident photons (anti-Stokes frequencies) is minor relative to that at frequencies lower than that of the incident photons (Stokes frequencies). Consequently, it is the Stokes frequencies that are usually analyzed. Therefore, the energy released from these molecules is specific to these molecules, and the specific molecules can be identified by detecting the released energy as electromagnetic waves.
It is also well known that, when specific molecules are irradiated with radiant energy in the form of electromagnetic waves, the molecules absorb the radiant energy to be excited in electronic excitation states, and generate fluorescence when the same return to ground states. Such fluorescence sharply reflects energy transfer, relaxation, reaction etc. in the excited states of the molecules, and hence the same is generally utilized as means for recognizing the dynamics of the molecules.
The inventors are making study on qualification and determination of specific molecules through a fluorescence/Raman spectrum.
In light which is generated from a sample, Rayleigh scattered light having the same wavelength as excitation light has a high intensity, while the intensity of Raman scattered light or fluorescence having a wavelength shifted from that of the excitation light is by far small as compared with the Rayleigh scattered light intensity. The intensity of Raman scattered light or fluorescence is proportionate to the concentration of a component causing Raman scattering or generating fluorescence in the sample, and hence the Raman scattered light or fluorescence is further weakened in case of measuring a biological substance of a small component or the like.
A laser unit is generally employed as a light source for the Raman spectrometry. The intensity of a laser beam outgoing from the laser unit is varied with time. Since the intensity of Raman scattered light is varied with intensity fluctuation of excitation light, the Raman spectrophotometry cannot be correctly performed unless a detected Raman scattered light intensity is corrected by the intensity of the excitation light.
Correction with respect to an excitation light intensity is made also in fluometry. While temporal fluctuation of a light source light intensity is reduced following improvement in accuracy of a constant current source in case of employing a xenon lamp as an excitation light source of fluorescence, correction with respect to the excitation light intensity is still necessary in order to make measurement in higher accuracy. When a dye laser is employed as an excitation light source of fluorescence, on the other hand, correction with respect to the excitation light intensity is indispensable.
In order to correct fluctuation of a light source intensity, excitation light is divided into a sample beam and a correction beam, for irradiating a sample with the sample beam and detecting fluorescence or Raman scattered light from light which is generated from the sample. On the other hand, the correction beam is detected by another photodetector, for correcting the detected value of the fluorescence or Raman scattered light by that of the correction beam in general. In this case, however, different photodetectors are required for detecting the fluorescence or Raman scattered light and the correction beam respectively, and hence the size of a measuring apparatus as well as the cost are increased.
In another correction method, Rayleigh scattered light from a sample is detected simultaneously with detection of Raman scattered light by a detector therefor, for correcting the intensity of the Raman scattered light on the basis of the Rayleigh scattered light intensity. In this case, the Raman scattered light and the Rayleigh scattered light can be detected by the same detector, whereby an apparatus structure is simplified and the cost is advantageously reduced. However, the Rayleigh scattered light is varied with the sample depending on the concentration of the sample, and hence the Raman spectrometry cannot be correctly performed.